Zinc-finger proteins with recognition regions that are engineered to bind to selected target sites are regularly linked to other zinc-finger proteins as well as to regulatory domains and used to modify gene expression and genomic target sites. For example, artificial nucleases comprising DNA binding domains operably linked to cleavage domains have been used for targeted alteration of genomic sequences, including, insertion of exogenous sequences, inactivation of one or more endogenous genes, creation of organisms (e.g., animal or crops) and cell lines with altered gene expression patterns, and the like. See, e.g., U.S. Patent Publication Nos. 20050064474; 20060063231; 20070134796; 20080015164 and International Publication No. 2007/139982.
Zinc-finger protein modules (e.g., engineered zinc fingers of one or more fingers) are typically linked to each other using “canonical” linker sequences of 5 amino acids such as TGEKP (SEQ ID NO:1) or longer flexible linkers. See, U.S. Pat. Nos. 6,479,626; 6,903,185; 7,153,949 and U.S. Patent Publication No. 20030119023. However, zinc-finger protein modules linked via these canonical linkers bind most effectively only when there is no gap between the linked module target subsites in the target nucleic acid molecule. Furthermore, previously-described long, flexible linkers designed to allow the linked modules to bind to target sites with 1, 2 or 3 base pair gaps do not distinguish between these different base pair gaps in terms of binding. See, U.S. Pat. Nos. 6,479,626; 6,903,185; 7,153,949 and U.S. Patent Publication No. 20030119023. Thus, there remains a need for methods and compositions for linking zinc-finger modules to each other that improves both the affinity of proteins that span a 1, 2, or 3 bp intermodule gap, as well improve the selectivity of these proteins for binding targets that span a gap of a desired length and do not bind non-selectively to other targets without the gap of that desired length. Linkers for zinc-finger modules that distinguish between 0, 1, 2, 3 or even more base pair gaps between adjacent module subsites would allow for greater design capability of any zinc-finger fusion proteins, including zinc-finger transcription factors (ZFP-TFs) and zinc finger nucleases (ZFNs).